CN111440802A - A sequence for expressing RP11-395G23.3 and its construct and construction method and application - Google Patents

Abstract

The present invention provides a sequence expressing RP11-395G23.3, which is shown in SEQ ID NO.1. The present invention also provides a construct, which comprises the DNA sequence shown in SEQ ID NO.1 between the upstream and downstream restriction sites of plasmid vector EcoRI and BamHI, and the construct sequence is SEQ ID NO.2 shown. In addition, the present invention also provides a method for preparing the construct: A. Obtainment of target gene fragments; B. Preparation of linearized expression vector; C. Construction of target gene into linearized expression vector; D. Preparation of DH5α competent cells and transformation; E, colony PCR identification of positive transformants; F, positive clones sent to sequencing. and the application of the construct in inhibiting endometrial cancer cells. The sequence for expressing RP11-395G23.3 and its construct of the present invention can effectively down-regulate the survival, migration and invasion capabilities of endometrial cancer cells, and have the effect of inhibiting endometrial cancer cells.

Description

A sequence for expressing RP11-395G23.3 and its construct and construction method with application

technical field

The present invention relates to the technical field of biology and genetic engineering, in particular to a sequence for expressing RP11-395G23.3 and its construct, construction method and application.

Background technique

Endometrial cancer is a group of malignant tumors of endometrial epithelial origin, and it is one of the most common malignant tumors in the female reproductive system, accounting for 20% to 30% of female reproductive tract malignant tumors. The incidence of endometrial cancer has increased and there is a younger trend. In my country, the incidence of endometrial cancer is second only to cervical cancer, and in developed countries such as Europe and the United States, its incidence rate ranks first in female genital tract malignant tumors. At present, the pathogenesis of endometrial cancer is still unclear, and it is mostly believed that the incidence is related to high estrogen stimulation and no progesterone antagonism. According to the different etiology, histology and biological characteristics, endometrial cancer can be divided into the following two subtypes: Type I endometrial cancer is estrogen-dependent and is the most common clinical type, accounting for the majority of endometrial cancer. 80%-90%. The histological type is mostly endometrial adenocarcinoma (75%-80%), and type II endometrial cancer is estrogen-independent, accounting for 10%-15% of endometrial cancer patients, and its histological type is mostly serous Papillary adenocarcinoma, a small part of which are clear cell carcinomas, with poorly differentiated cells and a high degree of malignancy. Surgical treatment of endometrial cancer is the first choice. On the basis of staging of surgical cases, adjuvant radiotherapy, chemotherapy and hormone therapy, but the therapeutic effect is not ideal.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to provide a sequence for expressing RP11-395G23.3 and its construct, construction method and application.

The technical scheme that the present invention takes for solving the technical problems existing in the known technology is:

The sequence for expressing RP11-395G23.3 of the present invention is shown in SEQ ID NO.1.

The construct of the present invention includes the DNA sequence shown in SEQ ID NO.1 between the upstream and downstream restriction sites of plasmid vectors EcoRI and BamHI, and the construct sequence is shown in SEQ ID NO.2.

The overexpression plasmid of RP11-395G23.3 is:

pLenti-EF1a-EGFP-F2A-Puro-CMV-LncRNA (RP11-395G23.3).

The plasmid vector is H145.

The construction method of the construct of the present invention comprises:

A. To obtain the target gene fragment, the target gene is obtained by amplification by PCR or by enzyme digestion;

Wherein, in the way of obtaining the target gene by PCR amplification, the target gene is amplified with the high-fidelity PrimeSTAR enzyme, and the reaction system and conditions are as follows:

Prepare PCR reaction solution according to the following components:

PCR reaction conditions were set as follows:

①3 Step method

②2 Step method

The PCR product was subjected to agarose gel electrophoresis to detect the amplification effect, and the target gene band was cut from the gel after agarose gel electrophoresis, and the gel was recovered with TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.3.0;

In the way of obtaining the target gene by enzyme digestion:

The plasmid containing the target gene was digested with restriction endonuclease. The digestion reaction system was: plasmid 2μg, 10x reaction buffer 5μL, restriction endonuclease 1μL, water to make up 50μL, incubate in 37℃ water bath for 2h above. The digestion product was subjected to agarose gel electrophoresis to detect the digestion effect, and the target gene band was cut from the gel after agarose gel electrophoresis, and the gel was recovered with TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0;

B. Preparation of the linearized expression vector. The expression vector was digested with restriction endonuclease. The enzyme digestion reaction system was: plasmid 2μg, 10x reaction Buffer 5μL, restriction endonuclease 1μL, water to make up 50μL, at 37 Incubate in a water bath for more than 2h. The digestion product was subjected to agarose gel electrophoresis to detect the digestion effect, and the target vector band was cut from the gel after agarose gel electrophoresis, and the gel was recovered with TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.3.0;

C. The target gene is constructed into the linearized expression vector, and the target gene is constructed into the linearized expression vector by any method in the seamless cloning kit, the seamless assembly kit and the use of T4 DNA ligase;

In the method of using the seamless cloning kit, the target gene fragment and the linearized vector are added to the centrifuge tube at a molar ratio of 2:1 to carry out the recombination reaction. The components are as follows:

The optimal amount of insert fragment used = [0.04×the number of bases in the insert fragment]ng(0.03pmol),

Optimum amount of linearized carrier used = [0.02×number of linearized carrier bases]ng(0.03pmol),

After mixing, incubate at 37°C for 30 minutes, then transfer to ice for 5 minutes. Direct transformation or store at -20°C and thaw transformation when transformation is required;

In the way of using the seamless assembly kit, the target gene fragment and the linearized vector are added to the centrifuge tube at a molar ratio of 1:1 to carry out the recombination reaction, and the groups are divided into:

The optimal amount of insert used = [0.02x base number of insert]ng (0.03pmol),

Optimum amount of linearized carrier used = [0.02×number of linearized carrier bases]ng(0.03pmol),

After mixing, incubate at 37°C for 30 minutes, then transfer to ice for 5 minutes. Direct transformation or store at -20°C and thaw transformation when transformation is required;

In the way of using T4DNA ligase:

The target gene fragment and the linearized vector were added to the centrifuge tube at a molar ratio of 3:1 to carry out the ligation reaction. The linearized expression vector is generally added with 100ng, and the target gene fragment is added in an amount of 300x the number of base pairs of the target gene/the number of base pairs of the linearized expression vector (unit: ng). The components are:

After mixing, ligate overnight at 16°C, and transfer 10 μL of the reaction liquid into competent cells;

D. Preparation and transformation of DH5α competent cells,

E. Colony PCR identifies positive transformants:

The transformants grown on the plate were picked and resuspended in 10 μL of LB medium, and 1 μL was used as a template for colony PCR identification. The reaction system and PCR cycle conditions were as follows:

The PCR reaction solution consists of:

PCR reaction conditions are:

3-step PCR

2-step PCR

F. Positive clones were sent for sequencing.

The forward sequencing primers for this construct are:

CMV-F CGCAAATGGGGCGGTAGGCGTG.

The reverse sequencing primers for this construct are:

WPRE-R CATAGCGTAAAAGGAGCAAACA.

Use of the constructs of the present invention in inhibiting endometrial cancer.

The advantages and positive effects that the present invention has are:

In the sequence for expressing RP11-395G23.3 of the present invention and its construct, overexpression of RP11-395G23.3 significantly reduced the activity, migration and invasion of HEC-1A and Ishikawa cells, and down-regulated the survival of endometrial cancer cells , migration and invasion ability; in addition, RP11-395G23.3 overexpression significantly induces EC cell apoptosis, and the dysregulation of RP11-395G23.3 is involved in the occurrence and development of endometrial cancer, and has the effect of inhibiting endometrial cancer cells.

Description of drawings

Figure 1 is a full spectrum of the constructs of the invention.

Detailed ways

The technical solutions of the present invention will be described in detail below through the accompanying drawings and specific embodiments.

The sequence for expressing RP11-395G23.3 of the present invention is shown in SEQ ID NO.1.

As shown in Figure 1, the construct of the present invention includes the DNA sequence shown in SEQ ID NO.1 between the upstream and downstream restriction sites of plasmid vector EcoRI and BamHI, and the construct sequence is shown in SEQ ID NO.2 As shown, the plasmid vector is H145.

The overexpression plasmid of RP11-395G23.3 is:

pLenti-EF1a-EGFP-F2A-Puro-CMV-LncRNA (RP11-395G23.3).

The construction method of the construct of the present invention comprises:

A. To obtain the target gene fragment, the target gene is obtained by amplification by PCR or by enzyme digestion;

Wherein, in the way of obtaining the target gene by PCR amplification, the target gene is amplified with the high-fidelity PrimeSTAR enzyme, and the reaction system and conditions are as follows:

Prepare PCR reaction solution according to the following components:

PCR reaction conditions were set as follows:

①3 Step method

②2 Step method

The PCR product was subjected to agarose gel electrophoresis to detect the amplification effect, and the target gene band was cut from the gel after agarose gel electrophoresis, and the gel was recovered with TaKaRa MiniBEST Agarose Gel DNAExtraction Kit Ver.3.0;

In the way of obtaining the target gene by enzyme digestion:

The plasmid containing the target gene was digested with restriction endonuclease. The digestion reaction system was: plasmid 2μg, 10x reaction buffer 5μL, restriction endonuclease 1μL, water to make up 50μL, incubate in 37℃ water bath for 2h above. The digestion product was subjected to agarose gel electrophoresis to detect the effect of digestion, and the target gene band was cut from the gel after agarose gel electrophoresis, and the gel was recovered with TaKaRa MiniBEST Agarose Gel DNAExtraction Kit Ver.4.0;

B. Preparation of the linearized expression vector. The expression vector was digested with restriction endonuclease. The enzyme digestion reaction system was: plasmid 2μg, 10x reaction Buffer 5μL, restriction endonuclease 1μL, water to make up 50μL, at 37 Incubate in a water bath for more than 2h. The digestion product was subjected to agarose gel electrophoresis to detect the digestion effect, and the target vector band was cut from the gel after agarose gel electrophoresis, and the gel was recovered with TaKaRa MiniBEST Agarose Gel DNAExtraction Kit Ver.3.0;

C. The target gene is constructed into the linearized expression vector, and the target gene is constructed into the linearized expression vector by any method in the seamless cloning kit, the seamless assembly kit and the use of T4 DNA ligase;

In the method of using the seamless cloning kit, the target gene fragment and the linearized vector are added to the centrifuge tube at a molar ratio of 2:1 to carry out the recombination reaction. The components are as follows:

The optimal amount of insert fragment used = [0.04×the number of bases in the insert fragment]ng(0.03pmol),

Optimum amount of linearized carrier used = [0.02×number of linearized carrier bases]ng(0.03pmol),

After mixing, incubate at 37°C for 30 minutes, then transfer to ice for 5 minutes. Direct transformation or store at -20°C and thaw transformation when transformation is required;

In the way of using the seamless assembly kit, the target gene fragment and the linearized vector are added to the centrifuge tube at a molar ratio of 1:1 to carry out the recombination reaction, and the groups are divided into:

The optimal amount of insert used = [0.02x base number of insert]ng (0.03pmol),

Optimum amount of linearized carrier used = [0.02×number of linearized carrier bases]ng(0.03pmol),

After mixing, incubate at 37°C for 30 minutes, then transfer to ice for 5 minutes. Direct transformation or store at -20°C and thaw transformation when transformation is required;

In the way of using T4DNA ligase:

The target gene fragment and the linearized vector were added to the centrifuge tube at a molar ratio of 3:1 to carry out the ligation reaction. The linearized expression vector is generally added with 100ng, and the target gene fragment is added in an amount of 300x the number of base pairs of the target gene/the number of base pairs of the linearized expression vector (unit: ng). The components are:

After mixing, ligate overnight at 16°C, and transfer 10 μL of the reaction liquid into competent cells;

D. Preparation and transformation of DH5α competent cells:

Take LB liquid medium LB/Amp (100ug/ml) solid petri dish, sterilize 0.1mol/l calcium chloride, deionized water, sterilize Ep tube, sterilize 0.1MCacl2/15% glycerol;

The preparation steps are:

1. Take DH5α bacteria to streak and cultivate overnight. (Scribing method: Dip a 100μL pipette tip in the bacterial solution, draw an S shape on the plate, start from the first corner for the second time, and divide it, and for the third time, draw a corner from the second line , gradually reduce the concentration of bacterial liquid);

2. Pick a round colony with a sterilized pipette tip in LB liquid medium (5mL in a test tube), 37°C, 200rpm, shake for about 12h, until the logarithmic growth phase;

3. Inoculate the strains in LB liquid medium (1mL/100mL) in a conical flask, 37 degrees, 200r/min, and shake for 2-3h until OD600=0.4. (You can read the words on the newspaper through the bacterial liquid in the conical flask, and it can be blurred and visible);

4A. Dispense into 2 50mL centrifuge tubes, centrifuge at 3000g at 4°C for 10min (pre-cool in centrifuge at 4°C before centrifugation), discard the supernatant;

5A. Add 10 mL of cold 0.1 MCacl2 to the centrifuge tube, resuspend the cell pellet, and shake on ice for 10 min;

6A. 4℃, 3000g, centrifuge for 10min, discard the supernatant;

7A. Add 10 mL of cold 0.1 MCacl2 to the centrifuge tube, resuspend the cell pellet, and shake on ice for 30 min;

8A. 4℃, 3000g, centrifuge for 10min, discard the supernatant;

9A. Add 2mL (2mL/50ml initial bacterial liquid volume) 0.1MCacl2/15% glycerol into a 50mL centrifuge tube, shake gently;

10A. Distribute 50 μL to 200 μL in each sterilized Ep tube, and store at -80°C;

4B. Add 1.5mL bacterial solution (2 tubes) to the Ep tube for 10 minutes on ice, 4000r/min at 0 to 4 degrees, and discard the supernatant for 2 minutes;

5B. Add 0.6 mL of 0.1 mol/L calcium chloride to ease the suspension, put on ice for 10 minutes, 4000 r/min for 10 min, and discard the supernatant;

6B. Add 0.2 mL of ice calcium chloride to slow down the suspended bacteria, divide into four tubes, and set aside on ice (store at -20 degrees);

convert

1. Put 0.5-2 μL of antibiotic-resistant plasmid into 100 μL of competent cells, and place on ice for 30 min;

2. Heat shock at 42°C for 90s, do not shake the tube, immediately put it on ice for 1-2 minutes;

3. Add 1mL LB liquid medium to the tube, 37℃, 200rpm, 30min～1h, activate the bacteria;

4. Take 50 bacteria liquid and apply it to the plate (if the transformation efficiency is low, you can take more bacteria liquid or centrifuge and discard the supernatant to remove the lower layer coating plate; after the bacteria coating stick is baked on the alcohol lamp, it should be left to cool for a while before coating the plate to prevent killing. (dead cells), when coating the plate, coat two competent cells, with resistance, no resistance, after transformation, apply one resistant bacterial solution, put the coated plate first and let it stand, wait for the bacterial solution to dry and then turn it upside down Incubate in a 37°C incubator;

E. Colony PCR identifies positive transformants:

The transformants grown on the plate were picked and resuspended in 10 μl of LB medium, and 1 μl was used as a template for colony PCR identification. The reaction system and PCR cycle conditions were as follows:

The PCR reaction solution consists of:

PCR reaction conditions are:

3-step PCR

2-step PCR

F. Positive clones were sent for sequencing.

Use of the constructs of the present invention in inhibiting endometrial cancer.

The regulation of RP11-395G23.3-miR-205-5p-PTEN on the activity, migration and invasion of endometrial cancer cells was detected, and the effect of RP11-395G23.3 on the proliferation of HEC-1A and Ishikawa cells was detected by CCK-8. The mobility of EC cells was evaluated by Transwell method and scratch method. Compared with the control group, overexpression of RP11-395G23.3 significantly reduced the viability, migration and invasion of HEC-1A and Ishikawa cells, and overexpression of miR-205-5p significantly increased the viability, migration and invasion of HEC-1A and Ishikawa cells , it can be seen that RP11-395G23.3 mediated the effects of miR-205-5p on EC cell viability, migration and invasion, and down-regulated the survival, migration and invasion of endometrial cancer cells. In addition, the effect of overexpression of RP11-395G23.3 on apoptosis of endometrial cancer cell lines was examined. The apoptosis of HEC-1A and Ishikawa cells was detected by flow cytometry. Compared with the control group, the overexpression of RP11-395G23.3 significantly induced the apoptosis of EC cells, and the overexpression of miR-205-5p significantly inhibited the apoptosis of EC cells. The occurrence and development of endometrial cancer has the effect of inhibiting endometrial cancer cells.

The above are only preferred embodiments of the present invention, and are not intended to limit the present invention in any form. Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Those skilled in the art, without departing from the scope of the technical solution of the present invention, will of course make some changes or modifications by using the disclosed technical content to become equivalent embodiments of equivalent changes, but any content that does not depart from the technical solution of the present invention, according to the present invention. Any simple modifications, equivalent changes and modifications made to the above embodiments by the technical essence of the invention fall within the scope of the technical solutions of the present invention.

sequence listing

<110> Obstetrics and Gynecology Hospital Affiliated to Fudan University

<120> A sequence for expressing RP11-395G23.3 and its construct and construction method and application

<141> 2020-03-14

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gagctcagcg ccggcgccgc gccgcccagc cccgccgaga ggggcgcact cgccgccgcg 180

gggcccgccg ccgctcaccg cagccccctc ctggcgaccc gcaagaggac tcagccgccg 240

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gggttcccaa atccaaaggg tccatctcca aggcgtcact ccaggggtcc gcggttcact 360

ttgctgcccc ttgcaggtga tgctgtgatt gttgcctgcg gactgggaag cacgatggct 420

ccaaaaggcc agcggggccg agaagcaggg acagagacca gcatttaagg gatctctggt 480

gggggcctcc ctggagttct ccacatgtaa atatagcgaa aaaagaaaac actacgttac 540

tagaataaac acgtcctaaa atttctgtgc aaaaacacct ttttgaacag caaagatttc 600

acccaccaaa acaaggaagc aagaagcttc cacagtgtgc aggatccgaa acaggaccgg 660

cccggcgttg ggtgagagtg ggggaggggt tgtcggcttc tccatccagc ttgccatgtt 720

ctgtcgtcgg tttcacattt tcagagagac tttaccagat gcaggaaatg aggatgaagg 780

caactctgga aggatgcaaa gaagggagag caagcactgt acctactgtc tgctgtctaa 840

gagggctggt ctcataacta aaaggtccag tccctgagat cttgtgtgca accgtcagaa 900

atagttcagg acatcaaatt gcacagagaa gattcagatg aagggaaaca agaactctgt 960

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cttttatggc tatgattttt ttcagtcatt attataaatc atgtgaaggt aaaataaatg 1260

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ttgaaatata ccctttgaaa tatgttgatt attctggaca tctgtaaggt gggagatttg 1200

aataaaaaga cttctgctgt cattattggc ttttatggct atgatttttt tcagtcatta 1260

ttataaatca tgtgaaggta aaataaatga tcattttagc cattctgttc tgcacagaat 1320

gctgttcaaa taaaacatca tctgatagga tcctcaacct ctggattaca aaatt 1375

Claims (6)

1. A sequence for expressing RP11-395G23.3, wherein the sequence is set forth in SEQ ID No. 1.

2. A construct is characterized by comprising a DNA sequence shown as SEQ ID NO.1 between restriction enzyme sites of EcoRI and BamHI of a plasmid vector, and the sequence of the construct is shown as SEQ ID NO. 2.

3. The construct of claim 2, wherein: the overexpression plasmid of RP11-395G23.3 is:

pLenti-EF1a-EGFP-F2A-Puro-CMV-LncRNA(RP11-395G23.3)。

4. the construct of claim 3, wherein: the plasmid vector was H145.

5. A method of constructing the construct of claim 2, comprising:

A. obtaining a target gene fragment, namely obtaining a target gene by a PCR amplification mode or an enzyme digestion mode;

in the mode of obtaining the target gene by PCR amplification, the high-fidelity PrimeSTAR enzyme is used for amplifying the target gene, and the reaction system and conditions are as follows:

the PCR reaction solution was prepared as follows:

the PCR conditions were set as follows:

① 3 Step method

② 2 Step method

Carrying out Agarose Gel electrophoresis on the PCR product to detect the amplification effect, cutting a target gene band from the Gel subjected to Agarose Gel electrophoresis, and recovering the Gel by using TaKaRa miniBEST Agarose Gel DNA Extraction Kit Ver.3.0;

in the mode of obtaining the target gene by enzyme digestion:

carrying out enzyme digestion on a plasmid containing a target gene by using restriction enzymes, wherein the enzyme digestion reaction system is that the plasmid is 2 mu g, 10x reaction Buffer is 5 mu L, the restriction enzymes are respectively 1 mu L, water is used for complementing 50 mu L, and the mixture is incubated in a water bath kettle at 37 ℃ for more than 2h, carrying out Agarose Gel electrophoresis on the enzyme digestion product to detect the enzyme digestion effect, cutting a target gene band from the Gel after the Agarose Gel electrophoresis, and using TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 as Gel for recovery;

B. preparing a linear expression vector, and carrying out enzyme digestion on the expression vector by using restriction enzymes, wherein the enzyme digestion reaction system is that 2 mu g of plasmid, 5 mu L of 10x reaction Buffer and 1 mu L of restriction enzymes respectively, supplementing 50 mu L of water, incubating for more than 2 hours in a water bath kettle at 37 ℃, carrying out Agarose Gel electrophoresis on the enzyme digestion product to detect the enzyme digestion effect, cutting a target vector strip from the Gel after the Agarose Gel electrophoresis, and using TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.3.0 for Gel recovery;

C. constructing a target gene into a linearized expression vector by using any one of a seamless cloning kit, a seamless assembly kit and T4DNA ligase;

in the mode of using the seamless cloning kit, a target gene fragment and a linearized vector are added into a centrifuge tube for recombination reaction according to the molar ratio of 2:1, and the components are as follows:

the total volume is 20 mu L

The optimum amount of the insert used was [0.04X number of bases of insert ] ng (0.03pmol),

the optimum amount of linearized vector used was [0.02x number of linearized vector bases ] ng (0.03pmol),

after mixing, the cells were incubated at 37 ℃ for 30 minutes, and then transferred to ice for 5 minutes. Directly transforming or unfreezing and transforming when being stored at-20 ℃ and the like;

in the mode of using the seamless assembly kit, the target gene fragment and the linearized vector are added into a centrifuge tube for recombination reaction according to the molar ratio of 1:1, and the recombination reaction is divided into the following groups:

the total volume is 20 mu L

The optimum amount of the insert used was [0.02X number of bases of insert ] ng (0.03pmol),

the optimum amount of linearized vector used was [0.02x number of linearized vector bases ] ng (0.03pmol),

after mixing, the cells were incubated at 37 ℃ for 30 minutes, and then transferred to ice for 5 minutes. Directly transforming or unfreezing and transforming when being stored at-20 ℃ and the like;

in the mode using T4DNA ligase:

adding the target gene fragment and the linearized vector into a centrifuge tube in a molar ratio of 3:1 for ligation reaction. Generally adding 100ng of linearized expression vector, wherein the addition amount of the target gene fragment is 300x of the base number of the target gene/the base number (unit ng) of the linearized expression vector, and each component is;

after mixing evenly, connecting overnight at 16 ℃, and taking 10 mu L reaction liquid to transform into competent cells;

D. preparing and transforming DH5 α competent cells;

E. colony PCR to identify positive transformants:

transformants grown on the plate were picked and resuspended in 10. mu.l of L B medium, and 1. mu.l was used as a template for colony PCR identification, and the reaction system and PCR cycling conditions were as follows:

the PCR reaction solution comprises the following components:

the PCR reaction conditions are as follows:

3 Step PCR

2 Step PCR

F. positive clones were sequenced.

6. Use of the construct of claim 2 for inhibiting endometrial cancer.

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